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Fig. S1. Expression pattern of Fgf10 in the limb-forming area and limb bud in the mouse embryo. (A-I) Fgf10 expression at E9.0 (A,D,G), E9.25 (B,E,H) and E9.5 (C,F,I) analyzed by whole-mount in situ hybridization. Expression in the whole embryo as viewed laterally (A-C), in the forelimb-forming area and forelimb bud as viewed dorsally (D-F), and in the hindlimb-forming area and hindlimb bud as viewed dorsally (G-I), is shown. The expression in the limb field and limb bud is indicated by arrowheads. Fgf10 expression is detected in the E9.0 forelimb field (D), becomes more intense at E9.25 (E) and E9.5 (F). Fgf10 expression in the hindlimb field becomes visible at E9.5 (I). he, head; im, intermediate mesoderm; t, tail bud.
Fig. S2. Successful inactivation of the Ctnnb1 gene in the mesenchyme by Hoxb6Cre in the hindlimb field at E9.5, and detailed morphology of the forelimb bud at E10.0. (A-D) Low magnification (A,B) and high magnification (C,D) of immunofluorescent images of β-catenin (green) and laminin (red) staining of the hindlimb-field of E9.5 embryos. Control (A,C) and Ctnnb1 cKO (B,D) embryos are shown. Laminin signal is detected in the basal membrane, and shows the boundary between the ectoderm and mesenchyme. The cKO embryos show significantly reduced mesenchymal β-catenin signal, whereas ectodermal signal is detected. (E,F) BATgal reporter activity in a control and Ctnnb1 cKO embryo at E9.5. A patchy signal of BATgal reporter activity is observed in the LPM of the hindlimb-forming area in the control embryo (E, black arrows), which is downregulated in the Ctnnb1 cKO embryo (F, red arrows). (G,H) Scanning electron microscopy images of E10.0 embryo forelimb buds. The Ctnnb1 cKO embryo exhibited a smaller forelimb bud (H, arrowheads) than the control littermate (Ctnnb1+/flox; Hoxb6Cre) embryo (G, arrows).
Fig. S3. T-cre; Ctnnb1flox/flox conditional knockout embryo. The control embryo (T-cre; Ctnnb1+/flox) has a well-developed body with both a forelimb (fl) and hindlimb bud (hl). The T-cre; Ctnnb1flox/flox conditional knockout embryo lacks all parts of its body posterior to the heart level, which precludes the possibility of limb initiation analysis studies. fl, forelimb bud; hl, hindlimb bud; he, heart.
Fig. S4. Fgf10 mutant analysis. Loss of Fgf10 does not affect proliferation and cell death in the LPM. Phospho-histone H3 (pHis3) and cell death were analyzed on sections prepared from the hindlimb-forming region of Fgf10−/− embryos and wild-type littermate embryos at E9.5. Statistical significance is examined by independent t-test. Loss of Fgf10 does not affect cell proliferation (pHis3) or cell death (TUNEL). Error bars indicate s.d.
Fig. S5. Normal expression pattern of Islet1 in the mouse embryo and successful inactivation of Islet1 in the hindlimb field of Islet1 cKO embryos. (A) The expression pattern of Islet1 is examined by whole-mount in situ hybridization from E8.5 to E10.5 in wild-type mouse embryos. The top panels show lateral views of the whole embryo. The middle panels show the hindlimb field and hindlimb bud, and the bottom panels show the forelimb field and forelimb bud, with anterior to the top. The expression in the hindlimb field and hindlimb bud are indicated by arrows. (B) Immunostaining of Islet1 in the control and Islet1 cKO embryo at E9.5. Islet1 signal is visible in the neural tube (arrows) and hindlimb field (arrows) in the control embryo. Although the signal in the neural tube is visible (arrows), the signal in the hindlimb field is not detected in the Islet1 cKO embryo.
